Abstract
Targeted gene modification has proved difficult in some model organisms, limiting their usefulness in studying basic biological functions. Different technologies based on naturally occurring modular DNA-binding proteins have been developed to rectify this issue. The most promising to date is the architecture of transcriptional activator like effectors nucleases (TALENs). TALEN proteins bind DNA utilizing arrays of repeat variable di-residue (RVD) modules, with each module interacting with a single target nucleotide. Each RVD module consists of about 33 nearly invariant amino acids, with the identity of positions 12 and 13 conferring binding specificity, so that RVD modules NI, NG, NN, and HD, bind to the nucleotide residues A, T, G, and C, respectfully. Artificial DNA binding domains consisting of arrays of these RVD modules can be used to target the non-specific FokI DNA nuclease domain to specific genomic targets where it induces dsDNA breaks (DSB). Error prone repair of DSBs can lead to the introduction of insertion or deletion mutations at the targeted site, ultimately resulting in frame shift mutations that can produce null alleles. TALEN RVD modules can be easily and very rapidly assembled using standard Golden Gate cloning methods. The 1:1 ratio simplifies both the assembly and design of TALENs and the use of standard molecular biology methods make the TALEN architecture amendable to use in Core Facilities. In the Zebrafish model system TALENs have been used to induce somatic mutations at targeted genomic sites with efficiencies reaching 100% of injected embryos and almost all the treated animals will have germ line mutations that can be stably transmitted. TALENs are being used to induce targeted mutations in Drosophila, C. elegans, and mammalian embryos as well as cultured cells, and it is likely the TALE-based DNA binding domains will soon be used routinely to manipulate gene expression in additional ways.